Purification of profibrinolysin



United States Patent F PURIFICATION OF PROFIBRINOLYSIN.

HeronO. Singher, Plainfield, and Jane M. Dressler, Somerville, N.J.,assignors to Ortho Pharmaceutical Corporation, a corporation of NewJersey No Drawing. Application January 10, 1958 Serial No. 708,060

9 Claims. (Cl. 167-65)- kinase, streptokinase, or by tissueextracts-fibronolysokinase, is converted into fibrinolysin (plasmin)which attacks the coagulation proteins. Inhibitors usually present withthe profibrinolysin retard this reaction. 1

According to the theory of Mullertz and Lassen (Pro- 2,922,745 1CPatented Jan. 26, 1960v through covalent linkages. Under appropriateexperimental conditions the nature of the binding can be controlled.Among other materials, heavy metal ions will form bonds of this type.Some heavy metal, protein compounds that are formed result in theprecipitation of the protein. Since the strength of binding (as measuredby the association constant) of the heavy metal ion to the protein is afunction of the nature of the bond, it is often possible to obtainmolecules whose association constants indicate more firm binding of thesame metal than possessed by the protein. These molecules (chelatingagents, exchange resins, etc.) can then be used to free the protein ofthe heavy metal. Similarly, if the physical conditions of the solutionare changed, the nature of the binding may be affected and theprecipitating agent removed.

Profibrinolysin is stable in highly acid solution (pH range-1 to 5)Whereas the same treatment denatures a very large number of otherbiologically active proteins.- Profibrinolysin is also soluble underthese conditions and so can be separated from insoluble contaminatingmate- I. rial. After this acid extract is neutralized (pH about ceedingsof the Society for Experimental Biology and 77 Medicine, 82, 264(1954)), streptokinase and presumably tissue kinases do notactivateprofibrinolysin directly but an inactive precursor of theactivator which would then convert profibrinolysin to fibrinolysin.Regardless, however, of the exact mechanism of fibrinolysis, it has beenshown that fibrin is aseptically dissolved by profibrinolysin activatedby streptokinase and streptodornase.

The intrathecal use of profibrinolysin activated by 'streptokinase tolyse tuberculosis exudates in vivo has been reported by Fletcher in theJournal of Clinical Investigations, 33, 1242-1251 (1954). Margulis hasdescribed the use of fibrinolysin to liquify blood clots in thirty-eightpatients (Archives of Surgery, 65, 511-521, 1952). Activatedprofibrinolysin also finds use in the debridement of wounds, burns, inthe treatment of chronic ulcers, and draining sinuses. v r 7 It is theobject of the present invention to prepare a highly active andconcentrated profibrinolysin.

It is another object of this invention to develop a process forpreparing profibrinolysin substantially free of inhibitors.

Many. procedures have been developed for the purification andpreparation of profibrinolysin. There are methods of Milstone,Christensen, Loomis, Klein, Cohn, Cliflton, etc. Manyof these proceduresdo not lend themselves readily to large scale processing, others yieldcrude products by modern standards and in still others the over allyields are low. Modern methods of plasma fractionation, such as Cohnsyield fractions that are mixtures, and result in a spreading of theactive components across many fractions. In order to obtain an activematerial in high yield of highly purified proenzyme from many of thesesources, it has been necessary to devise a new technique. We have nowdiscovered that profibrinolysin may be purified by adding to an aqueoussolution thereof, a water soluble salt of mercury, cadmium, Zinc or leadand precipitating under controlled conditions the mercury, cadmium,zinc, or lead salt of profibrinolysin.

Proteins can form compounds of either an ionic nature through ionicbonds or by the formation of complexes 6.0-8.0) 1 to 9 millimoles ofzinc ion per liter are added. This precipitates out the profibrinolysin.If the pH is less than about 6, impurities are co-precipitated with theprofibrinolysin salt. If the pH is greater than about 8, the yield ofthe precipitated profibrinolysin salt is re: duced.

The zinc salt may be further purified by alcohol frac tionation(precipitation from alcohol) or the zinc may be removed by dialysis, ionexchange, or chelation. The re-. sultant material may be precipitatedbythe addition of alcohol or it may be lyophilized. Other heavy metal ionswill also induce precipitation, specifically, mercury, cadmium and lead.The metal salt precipitate is then decomposed by compounds having agreater association constant with respect to the metal than plasminogen,or

the metal ions may be removed by dialysis.

The following examples are illustrative of the process and product ofthe present invention but are not to be construed as limiting.

EXAMPLE I Two hundred and thirty-four grams of Precipitate III; (made bymethods 6 and 9 of Cohn) which assayed-4000 units per milligram, isextracted with 10 volumes (2340 ml.) of 0.04 N sulfuric acid by'stirringfor 1 hour at 5 C. (pH 2.1). The insoluble material is removed bycentrifugation at 5 C., and the supernatant liquid is adjusted to pH4.4. The solution is again centrifuged at 5 C., and the residue isdiscarded. The pH of the supernatant is adjusted to 6.0 with 5 N sodiumhydroxide; the solution is again centrifuged at 5 C., and the residuediscarded; The supernatant is adjusted to pH 7.2 and the volume of thissupernatant is 2350 ml. To this solution is added 23.5 ml. of zincglycinate solution (about 17 millimoles of zinc ion). The pH is adjustedfrom 6.8 to 7.2 with 0.7 ml. of 1 N sodium hydroxide and the solution isallowed to stand overnight at 5 C., to assure complete precipitation ofthe zinc salt. The precipitate is isolated by centrifuging at 5 C., for30 minutes at 4000 r .p.m. The zinc-plasminogen precipitate weighs 7grams.

The zin'c-plasminogen precipitate is suspended in 7 ml.

resin is allowed to settle and the supernatant is poured off. The resinis washed by decantation threetimes with 10 m l-portions of water andthe wash solutions are-combined with the supernatant. The total volumeof the combined supernatant is adjusted to 50 ml. and the of thesupernatant is adjusted to 2.1 with 1 N hydrochloricacid.

A second 7-gram portion of Dowlex 50 resin (Hpform) is added'to thissupernatant and the suspension'is stirred at C., for 30 minutes. Thesupernatant is decanted and theresin is washed twice by decantation withwater as described above. The washings are added to the supernatant togive a combined volume of 70 ml.

A third 7-gram portion of Dowex 50 resin (H form) is added to thissupernatant and the suspension is stirred at 5 .C., for 30 minutes. Thesupernatant is decanted and the resin is washed twice by decantationwith water.

solution to 0.5 gram of fibrinogen and mix with a stirring rod in 37 C.water bath until the fibrinogen is The final volume of the combinedwashings and superof the human placenta and assaying about 500 units permilligram of dry weight was suspended in 10 ml. of 0.05

N sulphuric acid and centrifuged. The supernatant was adjusted to pH 4.5and again centrifuged The PIG? cipitate was discarded. This solution wasadjusted to pH 7 and 5 millimoles of zinc per liter added. Theprecipitate was removed and 8 milligrams of disodium ethylenediaminetetraacetic acid were added per gram of precipitate. This was dialyzedagainst distilledwater and the dialysate lyophilized. The resultantwhite powder had 23,000 units per milligram, and is free of thrombinsince a 1% solution will not form a clot with a 1% solution of humanfibrinolysin within 5 minutes when added thereto and incubated at 37 C.

' In determining the activity of the profibrinolysin derived accordingto Examples I and II, the following method was employed. The HouseStandard sample of profibrinolysin had an activity of 11,000 units permilligram of dry weight.

Profibrinolysin assay fibrin clot method A. SOLUTIONS dissolved. Keep at37 C. until used. Make fresh each day.

(6) Streptokinase: Dissolve a 20,000 unit vial in 4 milliliters ofsaline. Use 0.05 milliliter-250 units-per test. Make fresh each'day.

(7) Thrombin: Dissolve thrombin in sufficient saline to give 10 NationalInstitutes of Health units per 0.05

'milliliter. Use 0.05 milliliter per test. Make'fresh each day. I V

B. METHOD (1) Set up 10 pairs of photoelectric colorimeter tubes in arack and number 1 to 10 in duplicate.

(2) To pairs 1 to9, add'2 milliliters of phosphate saline butter.

(3) To pair 10, add 2.4 milliliters of phosphate saline buffer (negativecontrol).

' (4) To pair 1, add 0.4 milliliter of standard profi brinolysin. ('5)To pairs 2 to 9, add 0.4 milliliter of appropriate unknown.

. (6) Add 2.0 milliliters of fibrinogen gelatin to each tube.-

(1) Saline-09 percent: Dissolve 9 grams of sodium chloride CI. in 1000milliliters of distilled water.

(2) Standard profibrinolysinHouse Standard: Ten

- milligrams are weighed on an analytical balance and diluted to 1000milliliters with saline in a volumetric flask.- Make fresh each time.

(3) Unknown solutions:

of saline.

10 milligrams in 1000 milliliters 20 milligrams in 1000 milliliters ofsaline. Use 0.4 20 milligrams in 500 milliliters of saline. milli- 30milligrams in 500 milliliters of saline. liter 20 milligrams in 250milliliters of saline. per

10 milligrams in 100 milliliters of saline. test.

20 milligrams in 10000 milliliters of saline. 20 milligrams in 5000milliliters of saline.

fresh each time.

Make

from heat and stir in gelatin. Cool to 37 C. and ad just to mil1iliterswith distilled water. Add gelatin I (7) Add 0.1 milliliter of saline tothe'first tube of each pair.

(8) Add 0.05 milliliter of-streptokinase to the second tube of eachpair.

. (9) tubes placing a square of Parafilm over each tube and invertingthrice.

(10) Place racks with tubes in constant temperature water bath at 25 C.immersing to the level of the re- 1 agents. 7

(11) Have photoelectric colorimeter adjusted, wave length 660 mu. I

(12) Add 0.05 milliliter of thrombin solution to the secand tube of eachpair, one every minute in succession. Mix eachtube after addition. Starttiming from the addition to tube 1.

(13) Use first tube-of pair l0'to adjust sensitivity.

(14) Use first tube of each pair for zero adjustment and readtransmission before reading transmission ofthe second tube of the pair.

(15) Read-each pair one minute after the preceding pair and once everyten minutes until the second tube of each pair reads l00 percenttransmission or until 120 minutes have passed. Record time andtransmission for each reading.

C. CALCULATIONS /2 lysis: Subtract the first reading from 100 percentand divide the difference by 2 and add to the first reading. This valueis the percent transmission at k lysis. /2 lysis time: Make a lineargraph plotting transmission on the ordinate and time in minutes on theabscissa. Plot the readings on either sideof the calculated /2 lysis.Connect the points and read from the graph the time, "corresponding tothe calculated /2 lysis This is the /2 1 lysis time.

Use 2 millili H Employing the graph of ,6 House Standard (/2 lysis timeplotted on the ordinate against the units of activity per milligram ofHouse Standard profibrinolysin plotted on the abscissa), check the lysistime of the standard against the unknown. Read the unitage oil? thecurves for each $6 lysis time of the unknown.

Unitage 0.4

ml. of saline mgm. in solution =activity in units/mg. dry WeightAlthough human blood was used as the source of profibrinolysin in thepreceding examples, any other source of profibrinolysin from mammalianblood, such as bovine blood, maybe used and treated by the same process.

The process described is efiective in obtaining a pro lfibrinolysin freeof inhibitors.

What is claimed is:

1. A method of treating profibrinolysin which comprises extracting witha highly acid solution having a pH of about 1 to 5, neutralizing theacid extract to a pH of about 6 to 8, adding to the neutralized extractthe water soluble salt of a metal selected from the group consisting ofcadmium, mercury, lead and zinc, to precipitate the profibrinolysin as ametal salt, and separating the purified profibrinolysin by treating theprecipitate with an ion exchange resin having free acid groups.

2. A method of treating profibrinolysin which comprises extracting witha highly acid solution having a pH of about 1 to 5, neutralizing theacid extract to a pH range of about 6 to 8, adding to the neutralizedextract the water soluble salt of a metal selected from the groupconsisting of cadmium, mercury, lead and zinc, to precipitate theprofibrinolysin as a metal salt, and dialyzing the precipitate to removemetal ions from the purified profibrinolysin.

3. A method of treating profibrinolysin which comprises extracting witha highly acid solution having a pH of about 1 to 5, neutralizing theacid extract to a pH range of about 6 to 8, adding to the neutralizedextract the water soluble salt of a metal selected from the groupconsisting of cadmium, mercury, lead and zinc, to precipitate theprofibrinolysin as a metal salt, and recovering the purifiedprofibrinolysin by treating the precipitate with disodiumethylenediamine tetraacetic acid.

4. A method of purifying profibrinolysin which comprises suspending theprofibrinolysin in a highly acid solution having a pH of about 4.5,separating the acid solution from insoluble contaminating material,neutralizing the acid solution until the pH range is between 6 and 8,adding the water soluble salt of a bivalent metal selected from thegroup consisting of cadmium, mercury, lead and zinc to precipitate theprofibrinolysin from the solution and separating the profibrinolysinfrom the precipitating metal by treating the precipitate with an ionexchange resin having free acid groups.

5. A process according to claim 1 in which the water soluble metal saltis cadmium salt.

6. A process according to claim 1 in which the water soluble metal saltis a mercury salt. 7

7. A process according to claim 1 in which the water soluble metal saltis a lead salt.

8. A process according to claim 1 in which the water soluble metal saltis a zinc salt.

9. A purified highly active profibrinolysin, the activity being about700-fold that of the starting human serum, said profibrinolysin beingfree of thrombin and substantially free of inhibitors, 21 1% solution ofsaid profibrinolysin being incapable of clotting a 1% solution of humanfibrinogen within 5 minutes when added thereto and incubated at 37 C.

References Cited in the file of this patent UNITED STATES PATENTS CohnNov. 13, 1956 OTHER REFERENCES

1. A METHOD OF TREATING PROFIBRINOLYSIN WHICH COMPRISES EXTRACTING WITHA HIGHLY ACID SOLUTION HAVING A PH OF ABOUT 1 TO 5, NEUTRALIZING THEACID EXTRACT TO A PH OF ABOUT 6 TO 8, ADDING TO THE NEUTRALIZED EXTRACTTHE WATER SOLUBLE SALT OF A METAL SELECTED FROM THE GROUP CONSISTING OFCADMIUM, MERCURY, LEAD AND ZINC, TO PRECIPITATE THE PROFIBRINOLYSIN AS AMETAL SALT, AND SEPARATING THE PURIFIED PROFIBRINOLYSIN BY TREATING THEPRECIPITATE WITH AN ION EXCHANGE RESIN HAVING FREE ACID GROUPS.
 9. APURIFIED HIGHLY ACTIVE PROFIBRINOLYSIN, THE ACTIVITY BEING ABOUT700-FOLD THAT OF THE STARTING HUMAN SERUM, SAID PROFIBRINOLYSIN BEINGFREE OF THROMBIN AND SUBSTANTIALLY FREE OF INHIBITORS, A 1% SOLUTION OFSAID PROFIBRINOLYSIN BEING INCAPABLE OF CLOTTING A 1% SOLUTION OF HUMANFIBRINOGEN WITHIN 5 MINUTES WHE ADDED THERETO AND INCUBATED AT 37*C.